Because sequence variation in the human immunodeficiency virus (HIV-1) most likely results from inaccurate replication by reverse transcriptase, we are attempting to understand the mechanisms responsible for reverse transcriptase errors. We have shown that this enzyme is error-prone during both DNA- and RNA-templated replication, but that fidelity is several-fold higher with RNA than with DNA. The unequal error rates with RNA and DNA templates suggest that mistakes during minus- and plus-strand DNA synthesis may not contribute equally to the mutation rate of HIV-1. We have focused on template[unreadable]primer misalignment-mediated RT errors. Frameshift error rates at homopolymeric sites are affected by changes in the sequence flanking the runs, including single nucleotide differences in the single-stranded template strand and in the double-stranded primer region as many as six nucleotides distant from the hot spot. Both increases and decreases in frameshift fidelity are observed, and most correlate with concomitant increases or decreases in the probability that HIV-1 RT terminated processive synthesis within the run. Strong termination sites rarely correspond to dATP as the "incoming" substrate opposite template T, specific positions in the template[unreadable]primer stem are more important for termination than the sequence of the template ahead of the primer, and changing one nucleotide position in the template.primer stem alters termination at positions a few nucleotides distant. These results suggest that the HIV-1 RT distinguishes both base residues and positions in the template[unreadable]primer stem, and that contacts well-removed from the active site are important for fidelity. We will continue to focus on elucidating the mechanisms responsible for the inaccuracy of HIV-1 RT, in the hope that this will provide insights into the interaction of the enzyme's active site with its substrates that may be useful in designing RT-targeted drugs.